Colony-Forming Cell (CFC) or Colony-Forming Unit (CFU) Assays: The Basics

Learn the origins of the CFU/CFC assay, originally developed by Bradley and Metcalf in 1966 to identify mouse progenitor cells. Understand how the assay was adapted for human cells by Pike and Robinson in 1970 and expanded for erythroid progenitors and other cell types through the 1970s.

Mix together methylcellulose, serum, growth factors, and target cells in a tube to create approximately 3 milliliters of assay mixture. Dispense the reagent into duplicate 35 millimeter petri dishes, with 1 milliliter per dish.

Incubate the prepared dishes for seven to fourteen days depending on the cell type and species being cultured. Count the resulting colonies under an inverted microscope after the incubation period.

Understand that the colony-forming assay identifies cells by their differentiation ability, not proliferation. Colony form and color depend on the cell type, with larger colonies indicating more primitive progenitors, and differentiation is required for colony identification.

Recognize that CFU/CFC assays function as viability and differentiation potential assays for detecting primitive and rare hematopoietic cells. Apply the assay to determine effects of compounds on cell differentiation and maturation processes in basic research.

Consider the significant limitations of classic colony-forming assays including manual enumeration subjectivity, high technical skill requirements, high coefficient of variation, long incubation periods, lack of standards for validation, and operator intensity. Recognize these drawbacks preclude use as a potency assay for cell therapy products.